Glucosyltransferase enzymes for production of glucan polymers

ABSTRACT

Reaction solutions are disclosed herein comprising water, sucrose and a glucosyltransferase enzyme that synthesizes poly alpha-1,3-glucan. The glucosyltransferase enzyme can synthesize insoluble glucan polymer having at least 50% alpha-1,3 glycosidic linkages and a number average degree of polymerization of at least 100. Further disclosed are methods of using such glucosyltransferase enzymes to produce insoluble poly alpha-1,3-glucan.

This application is a divisional of pending application Ser. No.14/036,049, filed Sep. 25, 2013, which claims the benefit of U.S.Provisional Application Nos. 61/705,177; 61/705,178; 61/705,179;61/705,180 and 61/705,181, each filed Sep. 25, 2012. All of these priorapplications are incorporated herein by reference in their entirety.

FIELD OF INVENTION

The invention is in the field of enzyme catalysis. Specifically, thisinvention pertains to producing high molecular weight, insoluble polyalpha-1,3-glucan using a glucosyltransferase enzyme.

BACKGROUND

Driven by a desire to find new structural polysaccharides usingenzymatic syntheses or genetic engineering of microorganisms or planthosts, researchers have discovered polysaccharides that arebiodegradable and can be made economically from renewably sourcedfeedstocks. One such polysaccharide is poly alpha-1,3-glucan, a glucanpolymer characterized by having alpha-1,3-glycosidic linkages. Thispolymer has been isolated by contacting an aqueous solution of sucrosewith a glucosyltransferase (gtf) enzyme isolated from Streptococcussalivarius (Simpson et al., Microbiology 141:1451-1460, 1995). Filmsprepared from poly alpha-1,3-glucan tolerate temperatures up to 150° C.and provide an advantage over polymers obtained from beta-1,4-linkedpolysaccharides (Ogawa et al., Fiber Differentiation Methods 47:353-362,1980).

U.S. Pat. No. 7,000,000 disclosed the preparation of a polysaccharidefiber using an S. salivarius gtfJ enzyme. At least 50% of the hexoseunits within the polymer of this fiber were linked viaalpha-1,3-glycosidic linkages. S. salivarius gtfJ enzyme utilizessucrose as a substrate in a polymerization reaction producing polyalpha-1,3-glucan and fructose as end-products (Simpson et al., 1995).The disclosed polymer formed a liquid crystalline solution when it wasdissolved above a critical concentration in a solvent or in a mixturecomprising a solvent. Continous, strong, cotton-like fibers wereobtained from this solution that could be spun and used in textileapplications.

Not all glucosyltransferase enzymes can produce glucan with a molecularweight and percentage of alpha-1,3 glycosidic linkages suitable for usein spinning fibers. For example, most glucosyltransferase enzymes do notproduce glucan having at least 50% alpha-1,3 glycosidic linkages and anumber average degree of polymerization of at least 100. Therefore, itis desirable to identify glucosyltransferase enzymes that can convertsucrose to glucan polymers having a high percentage of alpha-1,3glycosidic linkages and high molecular weight.

SUMMARY OF INVENTION

In one embodiment, the invention concerns a reaction solution comprisingwater, sucrose and a glucosyltransferase enzyme that synthesizes polyalpha-1,3-glucan. The glucosyltransferase enzyme comprises an amino acidsequence that is at least 90% identical to the amino acid sequence ofSEQ ID NO:4, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:20, SEQID NO:26, SEQ ID NO:28, SEQ ID NO:30, or SEQ ID NO:34.

In a second embodiment, the glucosyltransferase enzyme in the reactionsolution synthesizes poly alpha-1,3-glucan having at least 50% alpha-1,3glycosidic linkages and a number average degree of polymerization of atleast 100. In a third embodiment, the glucosyltransferase synthesizespoly alpha-1,3-glucan having 100% alpha-1,3 glycosidic linkages and anumber average degree of polymerization of at least 100. In a fourthembodiment, the glucosyltransferase enzyme synthesizes polyalpha-1,3-glucan having 100% alpha-1,3 glycosidic linkages and a numberaverage degree of polymerization of at least 250.

In a fifth embodiment, the reaction solution comprises a primer. In asixth embodiment, this primer can be dextran or hydrolyzed glucan.

In a seventh embodiment, the invention concerns a method for producingpoly alpha-1,3-glucan comprising the step of contacting at least water,sucrose, and a glucosyltransferase enzyme that synthesizes polyalpha-1,3-glucan. The glucosyltransferase enzyme comprises an amino acidsequence that is at least 90% identical to the amino acid sequence ofSEQ ID NO:4, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:20, SEQID NO:26, SEQ ID NO:28, SEQ ID NO:30, or SEQ ID NO:34. The polyalpha-1,3-glucan produced in this method can optionally be isolated.

In an eighth embodiment, the glucosyltransferase enzyme used in themethod synthesizes poly alpha-1,3-glucan having at least 50% alpha-1,3glycosidic linkages and a number average degree of polymerization of atleast 100. In a ninth embodiment, the glucosyltransferase enzymesynthesizes poly alpha-1,3-glucan having 100% alpha-1,3 glycosidiclinkages and a number average degree of polymerization of at least 100.In a tenth embodiment, the glucosyltransferase enzyme synthesizes polyalpha-1,3-glucan having 100% alpha-1,3 glycosidic linkages and a numberaverage degree of polymerization of at least 250.

In an eleventh embodiment, the contacting step of the method furthercomprises contacting a primer with the water, sucrose, andglucosyltransferase enzyme. In a twelfth embodiment, this primer can bedextran or hydrolyzed glucan.

BRIEF DESCRIPTION OF THE SEQUENCES

TABLE 1 Summary of Nucleic Acid and Protein SEQ ID Numbers Nucleic acidProtein SEQ SEQ Description ID NO. ID NO. “0874 gtf”, Streptococcussobrinus. DNA codon- 1  2 optimized for expression in E. coli. The first156 (1435 aa) amino acids of the protein are deleted compared to GENBANKIdentification No. 450874, which discloses “glucosyltransferase-I”.“6855 gtf”, Streptococcus salivarius SK126. DNA 3  4 codon-optimized forexpression in E. coli. The first (1341 aa) 178 amino acids of theprotein are deleted compared to GENBANK Identification No. 228476855,which discloses “glucosyltransferase-SI”. “2379 gtf”, Streptococcussalivarius. DNA codon- 5  6 optimized for expression in E. coli. Thefirst 203 (1247 aa) amino acids of the protein are deleted compared toGENBANK Identification No. 662379, which discloses“glucosyltransferase”. “7527” or “gtfJ”, Streptococcus salivarius. DNA 7 8 codon-optimized for expression in E. coli. The first 42 (1477 aa)amino acids of the protein are deleted compared to GENBANKIdentification No. 47527, which discloses “glucosyltransferase-I”. “1724gtf”, Streptococcus downei. DNA codon- 9 10 optimized for expression inE. coli. The first 162 (1436 aa) amino acids of the protein are deletedcompared to GENBANK Identification No. 121724, which discloses“glucosyltransferase-I”. “0544 gtf”, Streptococcus mutans. DNA codon- 1112 optimized for expression in E. coli. The first 164 (1313 aa) aminoacids of the protein are deleted compared to GENBANK Identification No.290580544, which discloses “glucosyltransferase-I”. “5926 gtf”,Streptococcus dentirousetti. DNA codon- 13 14 optimized for expressionin E. coli. The first 144 (1323 aa) amino acids of the protein aredeleted compared to GENBANK Identification No. 167735926, whichdiscloses “glucosyltransferase-I”. “4297 gtf”, Streptococcus oralis. DNAcodon- 15 16 optimized for expression in E. coli. The first 228 (1348aa) amino acids of the protein are deleted compared to GENBANKIdentification No. 7684297, which discloses “glucosyltransferase”. “5618gtf”, Streptococcus sanguinis. DNA codon- 17 18 optimized for expressionin E. coli. The first 223 (1348 aa) amino acids of the protein aredeleted compared to GENBANK Identification No. 328945618, whichdiscloses “glucosyltransferase-S”. “2765 gtf”, unknown Streptococcus sp.C150. DNA 19 20 codon-optimized for expression in E. coli. The first(1340 aa) 193 amino acids of the protein are deleted compared to GENBANKIdentification No. 322372765, which discloses “glucosyltransferase-S”.“4700 gtf”, Leuconostoc mesenteroides. DNA codon- 21 22 optimized forexpression in E. coli. The first 36 amino (1492 aa) acids of the proteinare deleted compared to GENBANK Identification No. 21654700, whichdiscloses “dextransucrase DsrD”. “1366 gtf”, Streptococcus criceti. DNAcodon- 23 24 optimized for expression in E. coli. The first 139 (1323aa) amino acids of the protein are deleted compared to GENBANKIdentification No. 146741366, which discloses “glucosyltransferase”.“0427 gtf”, Streptococcus sobrinus. DNA codon- 25 26 optimized forexpression in E. coli. The first 156 (1435 aa) amino acids of theprotein are deleted compared to GENBANK Identification No. 940427, whichdiscloses “GTF-I”. “2919 gtf”, Streptococcus salivarius PS4. DNA 27 28codon-optimized for expression in E. coli. The first 92 (1340 aa) aminoacids of the protein are deleted compared to GENBANK Identification No.383282919, which discloses “putative glucosyltransferase”. “2678 gtf”,Streptococcus salivarius K12. DNA codon- 29 30 optimized for expressionin E. coli. The first 188 (1341 aa) amino acids of the protein aredeleted compared to GENBANK Identification No. 400182678, whichdiscloses “dextransucrase-S”. “2381 gtf”, Streptococcus salivarius. DNAcodon- 31 32 optimized for expression in E. coli. The first 273 (1305aa) amino acids of the protein are deleted compared to GENBANKIdentification No. 662381, which discloses “glucosyltransferase”. “3929gtf”, Streptococcus salivarius JIM8777. DNA 33 34 codon-optimized forexpression in E. coli. The first (1341 aa) 178 amino acids of theprotein are deleted compared to GENBANK Identification No. 387783929,which discloses “glucosyltransferase-S precursor (GTF-S)(Dextransucrase) (Sucrose 6-glucosyltransferase)”. “6907 gtf”,Streptococcus salivarius SK126. DNA 35 36 codon-optimized for expressionin E. coli. The first (1331 aa) 161 amino acids of the protein aredeleted compared to GENBANK Identification No. 228476907, whichdiscloses “glucosyltransferase-SI”. “6661 gtf”, Streptococcus salivariusSK126. DNA 37 38 codon-optimized for expression in E. coli. The first(1305 aa) 265 amino acids of the protein are deleted compared to GENBANKIdentification No. 228476661, which discloses “glucosyltransferase-SI”.“0339 gtf”, Streptococcus gallolyticus ATCC 43143. 39 40 DNAcodon-optimized for expression in E. coli. The (1310 aa) first 213 aminoacids of the protein are deleted compared to GENBANK Identification No.334280339, which discloses “glucosyltransferase”. “0088 gtf”,Streptococcus mutans. DNA codon- 41 42 optimized for expression in E.coli. The first 189 (1267 aa) amino acids of the protein are deletedcompared to GENBANK Identification No. 3130088, which discloses“glucosyltransferase-SI”. “9358 gtf”, Streptococcus mutans UA159. DNA 4344 codon-optimized for expression in E. coli. The first (1287 aa) 176amino acids of the protein are deleted compared to GENBANKIdentification No. 24379358, which discloses “glucosyltransferase-S”.“8242 gtf”, Streptococcus gallolyticus ATCC BAA- 45 46 2069. DNAcodon-optimized for expression in E. coli. (1355 aa) The first 191 aminoacids of the protein are deleted compared to GENBANK Identification No.325978242, which discloses “glucosyltransferase-I”. “3442 gtf”,Streptococcus sanguinis SK405. DNA 47 48 codon-optimized for expressionin E. coli. The first (1348 aa) 228 amino acids of the protein aredeleted compared to GENBANK Identification No. 324993442, whichdiscloses a “ . . . signal domain protein”. “7528 gtf”, Streptococcussalivarius. DNA codon- 49 50 optimized for expression in E. coli. Thefirst 173 (1427 aa) amino acids of the protein are deleted compared toGENBANK Identification No. 47528, which discloses “glucosyltransferaseS”. “3279 gtf”, Streptococcus sp. C150. DNA codon- 51 52 optimized forexpression in E. coli. The first 178 (1393 aa) amino acids of theprotein are deleted compared to GENBANK Identification No. 322373279,which discloses “glucosyltransferase S”. “6491 gtf”, Leuconostoc citreumKM20. DNA codon- 53 54 optimized for expression in E. coli. The first244 (1262 aa) amino acids of the protein are deleted compared to GENBANKIdentification No. 170016491, which discloses “glucosyltransferase”.“6889 gtf”, Streptococcus salivarius SK126. DNA 55 56 codon-optimizedfor expression in E. coli. The first (1427 aa) 173 amino acids of theprotein are deleted compared to GENBANK Identification No. 228476889,which discloses “glucosyltransferase-I”. “4154 gtf”, Lactobacillusreuteri. DNA codon- 57 58 optimized for expression in E. coli. The first38 amino (1735 aa) acids of the protein are deleted compared to GENBANKIdentification No. 51574154, which discloses “glucansucrase”. “3298gtf”, Streptococcus sp. C150. The first 209 59 amino acids of theprotein are deleted compared to (1242 aa) GENBANK Identification No.322373298, which discloses “glucosyltransferase-S”. “Wild type gtfJ”,Streptococcus salivarius. 60 GENBANK Identification No. 47527. (1518 aa)Wild type gtf corresponding to 2678 gtf, Streptococcus 61 salivariusK12. GENBANK Identification No. (1528 aa) 400182678, which discloses“dextransucrase-S”. Wild type gtf corresponding to 6855 gtf,Streptococcus 62 salivarius SK126. GENBANK Identification No. (1518 aa)228476855, which discloses “glucosyltransferase-SI”. Wild type gtfcorresponding to 2919 gtf, Streptococcus 63 salivarius PS4. GENBANKIdentification No. (1431 aa) 383282919, which discloses “putativeglucosyltransferase”. Wild type gtf corresponding to 2765 gtf,Streptococcus 64 sp. C150. GENBANK Identification No. 322372765, (1532aa) which discloses “glucosyltransferase-S”.

DETAILED DESCRIPTION OF THE INVENTION

The disclosures of all cited patent and non-patent literature areincorporated herein by reference in their entirety.

As used herein, the term “invention” or “disclosed invention” is notmeant to be limiting, but applies generally to any of the inventionsdefined in the claims or described herein. These terms are usedinterchangeably herein.

The terms “poly alpha-1,3-glucan”, “alpha-1,3-glucan polymer” and“glucan polymer” are used interchangeably herein. Poly alpha-1,3-glucanis a polymer comprising glucose monomeric units linked together byglycosidic linkages, wherein at least about 50% of the glycosidiclinkages are alpha-1,3-glycosidic linkages. Poly alpha-1,3-glucan is atype of polysaccharide. The structure of poly alpha-1,3-glucan can beillustrated as follows:

The terms “glycosidic linkage” and “glycosidic bond” are usedinterchangeably herein and refer to the type of covalent bond that joinsa carbohydrate (sugar) molecule to another group such as anothercarbohydrate. The term “alpha-1,3-glycosidic linkage” as used hereinrefers to the type of covalent bond that joins alpha-D-glucose moleculesto each other through carbons 1 and 3 on adjacent alpha-D-glucose rings.This linkage is illustrated in the poly alpha-1,3-glucan structureprovided above. Herein, “alpha-D-glucose” will be referred to as“glucose”.

The term “sucrose” herein refers to a non-reducing disaccharide composedof an alpha-D-glucose molecule and a beta-D-fructose molecule linked byan alpha-1,2-glycosidic bond. Sucrose is known commonly as table sugar.

The “molecular weight” of the poly alpha-1,3-glucan herein can berepresented as number-average molecular weight (M_(n)) or asweight-average molecular weight (M_(w)). Alternatively, molecular weightcan be represented as Daltons, grams/mole, DPw (weight average degree ofpolymerization), or DPn (number average degree of polymerization).Various means are known in the art for calculating these molecularweight measurements such as with high-pressure liquid chromatography(HPLC), size exclusion chromatography (SEC), or gel permeationchromatography (GPC).

The terms “glucosyltransferase enzyme”, “gtf enzyme”, “gtf enzymecatalyst”, “gtf”, and “glucansucrase” are used interchangeably herein.The activity of a gtf enzyme herein catalyzes the reaction of thesubstrate sucrose to make the products poly alpha-1,3-glucan andfructose. Other products (byproducts) of a gtf reaction can includeglucose (where glucose is hydrolyzed from the glucosyl-gtf enzymeintermediate complex), various soluble oligosaccharides (DP2-DP7), andleucrose (where glucose of the glucosyl-gtf enzyme intermediate complexis linked to fructose). Leucrose is a disaccharide composed of glucoseand fructose linked by an alpha-1,5 linkage. Wild type forms ofglucosyltransferase enzymes generally contain (in the N-terminal toC-terminal direction) a signal peptide, a variable domain, a catalyticdomain, and a glucan-binding domain. A gtf herein is classified underthe glycoside hydrolase family 70 (GH70) according to the CAZy(Carbohydrate-Active EnZymes) database (Cantarel et al., Nucleic AcidsRes. 37:D233-238, 2009).

The terms “reaction” and “enzymatic reaction” are used interchangeablyherein and refer to a reaction that is performed by aglucosyltransferase enzyme. A “reaction solution” as used hereingenerally refers to a solution comprising at least one activeglucosyltransferase enzyme in a solution comprising sucrose and water,and optionally other components. It is in the reaction solution wherethe step of contacting water, sucrose and a glucosyltransferase enzymeis performed. The term “under suitable reaction conditions” as usedherein, refers to reaction conditions that support conversion of sucroseto poly alpha-1,3-glucan via glucosyltransferase enzyme activity. Thereaction herein is not naturally occurring.

The terms “percent by volume”, “volume percent”, “vol %” and “v/v %” areused interchangeably herein. The percent by volume of a solute in asolution can be determined using the formula: [(volume ofsolute)/(volume of solution)]×100%.

The terms “percent by weight”, “weight percentage (wt %)” and“weight-weight percentage (% w/w)” are used interchangeably herein.Percent by weight refers to the percentage of a material on a mass basisas it is comprised in a composition, mixture, or solution.

The terms “increased”, “enhanced” and “improved” are usedinterchangeably herein. These terms refer to a greater quantity oractivity such as a quantity or activity slightly greater than theoriginal quantity or activity, or a quantity or activity in large excesscompared to the original quantity or activity, and including allquantities or activities in between. Alternatively, these terms mayrefer to, for example, a quantity or activity that is at least 1%, 2%,3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%,19% or 20% more than the quantity or activity for which the increasedquantity or activity is being compared.

The terms “polynucleotide”, “polynucleotide sequence”, and “nucleic acidsequence” are used interchangeably herein. These terms encompassnucleotide sequences and the like. A polynucleotide may be a polymer ofDNA or RNA that is single- or double-stranded, that optionally containssynthetic, non-natural or altered nucleotide bases. A polynucleotide maybe comprised of one or more segments of cDNA, genomic DNA, syntheticDNA, or mixtures thereof.

The term “gene” as used herein refers to a polynucleotide sequence thatexpresses a protein, and which may refer to the coding region alone ormay include regulatory sequences upstream and/or downstream to thecoding region (e.g., 5′ untranslated regions upstream of thetranscription start site of the coding region). A gene that is “native”or “endogenous” refers to a gene as found in nature with its ownregulatory sequences; this gene is located in its natural location inthe genome of an organism. “Chimeric gene” refers to any gene that isnot a native gene, comprising regulatory and coding sequences that arenot found together in nature. A “foreign” or “heterologous” gene refersto a gene that is introduced into the host organism by gene transfer.Foreign genes can comprise native genes inserted into a non-nativeorganism, native genes introduced into a new location within the nativehost, or chimeric genes. The polynucleotide sequences in certainembodiments disclosed herein are heterologous. A “transgene” is a genethat has been introduced into the genome by a transformation procedure.A “codon-optimized gene” is a gene having its frequency of codon usagedesigned to mimic the frequency of preferred codon usage of the hostcell.

A native amino acid sequence or polynucleotide sequence is naturallyoccurring, whereas a non-native amino acid sequence or polynucleotidesequence does not occur in nature.

“Coding sequence” as used herein refers to a DNA sequence that codes fora specific amino acid sequence. “Regulatory sequences” as used hereinrefer to nucleotide sequences located upstream of the coding sequence'stranscription start site, 5′ untranslated regions and 3′ non-codingregions, and which may influence the transcription, RNA processing orstability, or translation of the associated coding sequence. Regulatorysequences may include promoters, enhancers, silencers, 5′ untranslatedleader sequence, introns, polyadenylation recognition sequences, RNAprocessing sites, effector binding sites, stem-loop structures and otherelements involved in regulation of gene expression.

The term “recombinant” as used herein refers to an artificialcombination of two otherwise separated segments of sequence, e.g., bychemical synthesis or by the manipulation of isolated segments ofnucleic acids by genetic engineering techniques. The terms“recombinant”, “transgenic”, “transformed”, “engineered” or “modifiedfor exogenous gene expression” are used interchangeably herein.

The term “transformation” as used in certain embodiments refers to thetransfer of a nucleic acid molecule into a host organism. The nucleicacid molecule may be a plasmid that replicates autonomously, or it mayintegrate into the genome of the host organism. Host organismscontaining the transformed nucleic acid fragments are referred to as“transgenic” or “recombinant” or “transformed” organisms or“transformants”.

The term “recombinant” or “heterologous” refers to an artificialcombination of two otherwise separate segments of sequence, e.g., bychemical synthesis or by the manipulation of isolated segments ofnucleic acids by genetic engineering techniques.

The terms “sequence identity” or “identity” as used herein with respectto polynucleotide or polypeptide sequences refer to the nucleic acidbases or amino acid residues in two sequences that are the same whenaligned for maximum correspondence over a specified comparison window.Thus, “percentage of sequence identity” or “percent identity” refers tothe value determined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide orpolypeptide sequence in the comparison window may comprise additions ordeletions (i.e., gaps) as compared to the reference sequence (which doesnot comprise additions or deletions) for optimal alignment of the twosequences. The percentage is calculated by determining the number ofpositions at which the identical nucleic acid base or amino acid residueoccurs in both sequences to yield the number of matched positions,dividing the number of matched positions by the total number ofpositions in the window of comparison and multiplying the results by 100to yield the percentage of sequence identity.

The Basic Local Alignment Search Tool (BLAST) algorithm, which isavailable online at the National Center for Biotechnology Information(NCBI) website, may be used, for example, to measure percent identitybetween or among two or more of the polynucleotide sequences (BLASTNalgorithm) or polypeptide sequences (BLASTP algorithm) disclosed herein.Alternatively, percent identity between sequences may be performed usinga Clustal algorithm (e.g., ClustalW or ClustalV). For multiplealignments using a Clustal method of alignment, the default values maycorrespond to GAP PENALTY=10 and GAP LENGTH PENALTY=10. Defaultparameters for pairwise alignments and calculation of percent identityof protein sequences using a Clustal method may be KTUPLE=1, GAPPENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. For nucleic acids, theseparameters may be KTUPLE=2, GAP PENALTY=5, WINDOW=4 and DIAGONALSSAVED=4. Alternatively still, percent identity between sequences may beperformed using an EMBOSS algorithm (e.g., needle) with parameters suchas GAP OPEN=10, GAP EXTEND=0.5, END GAP PENALTY=false, END GAP OPEN=10,END GAP EXTEND=0.5 using a BLOSUM matrix (e.g., BLOSUM62).

Various polypeptide amino acid sequences and polynucleotide sequencesare disclosed herein as features of certain embodiments of the disclosedinvention. Variants of these sequences that are at least about 70-85%,85-90%, or 90%-95% identical to the sequences disclosed herein can beused. Alternatively, a variant amino acid sequence or polynucleotidesequence can have at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% identity with a sequence disclosedherein. The variant amino acid sequence or polynucleotide sequence hasthe same function/activity of the disclosed sequence, or at least about85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% of the function/activity of the disclosed sequence.

The term “isolated” as used in certain embodiments refers to anycellular component that has been completely or partially purified fromits native source (e.g., an isolated polynucleotide or polypeptidemolecule). In some instances, an isolated polynucleotide or polypeptidemolecule is part of a greater composition, buffer system or reagent mix.For example, the isolated polynucleotide or polypeptide molecule can becomprised within a cell or organism in a heterologous manner. Anotherexample is an isolated glucosyltransferase enzyme.

Embodiments of the disclosed invention concern a reaction solutioncomprising water, sucrose and a glucosyltransferase enzyme thatsynthesizes poly alpha-1,3-glucan. The glucosyltransferase enzymecomprises an amino acid sequence that is at least 90% identical to theamino acid sequence of SEQ ID NO:4, SEQ ID NO:10, SEQ ID NO:12, SEQ IDNO:14, SEQ ID NO:20, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, or SEQ IDNO:34. Significantly, these glucosyltransferase enzymes can synthesizepoly alpha-1,3-glucan having at least 50% alpha-1,3 glycosidic linkagesand a number average degree of polymerization of at least 100. Suchglucan is suitable for use in spinning fibers and in other industrialapplications.

The molecular weight of the poly alpha-1,3-glucan produced by theglucosyltransferase enzymes herein can be measured as DP_(n) (numberaverage degree of polymerization). Alternatively, the molecular weightof the poly alpha-1,3-glucan can be measured in terms of Daltons,grams/mole, or as DP_(w) (weight average degree of polymerization). Thepoly alpha-1,3-glucan in certain embodiments of the invention can have amolecular weight in DP_(n) or DP_(w) of at least about 100. Themolecular weight of the poly alpha-1,3-glucan can alternatively be atleast about 250 DP_(n) or DP_(w). Alternatively still, the DP_(n) orDP_(w) of the poly alpha-1,3-glucan can be at least about 100, 150, 200,250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900,950, or 1000 (or any integer between 100 and 1000).

The molecular weight of the poly alpha-1,3-glucan herein can be measuredusing any of several means known in the art. For example, glucan polymermolecular weight can be measured using high-pressure liquidchromatography (HPLC), size exclusion chromatography (SEC), or gelpermeation chromatography (GPC).

The poly alpha-1,3-glucan herein is preferably linear/unbranched. Thepercentage of glycosidic linkages between the glucose monomer units ofthe poly alpha-1,3-glucan that are alpha-1,3 is at least about 50%, 60%,70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In such embodiments,accordingly, the poly alpha-1,3-glucan has less than about 50%, 40%,30%, 20%, 10%, 5%, 4%, 3%, 2%, 1%, or 0% of glycosidic linkages that arenot alpha-1,3.

It is understood that the higher the percentage of alpha-1,3-glycosidiclinkages present in the poly alpha-1,3-glucan, the greater theprobability that the poly alpha-1,3-glucan is linear, since there arelower occurrences of certain glycosidic linkages forming branch pointsin the polymer. In certain embodiments, the poly alpha-1,3-glucan has nobranch points or less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or1% branch points as a percent of the glycosidic linkages in the polymer.Examples of branch points include alpha-1,6 branch points, such as thosethat are present in mutan polymer.

The glycosidic linkage profile of the poly alpha-1,3-glucan can bedetermined using any method known in the art. For example, the linkageprofile can be determined using methods that use nuclear magneticresonance (NMR) spectroscopy (e.g., ¹³C NMR or ¹H NMR). These and othermethods that can be used are disclosed in Food Carbohydrates: Chemistry,Physical Properties, and Applications (S. W. Cui, Ed., Chapter 3, S. W.Cui, Structural Analysis of Polysaccharides, Taylor & Francis Group LLC,Boca Raton, Fla., 2005), which is incorporated herein by reference.

The poly alpha-1,3-glucan herein may be characterized by any combinationof the aforementioned percentages of alpha-1,3 linkages and molecularweights. For example, the poly alpha-1,3-glucan produced in a reactionsolution herein can have at least 50% alpha-1,3 glycosidic linkages anda DP_(n) or DP_(w) of at least 100. As another example, the polyalpha-1,3-glucan can have 100% alpha-1,3 glycosidic linkages and aDP_(n) or DP_(w) of at least 100. The poly alpha-1,3-glucan in stillanother example can have 100% alpha-1,3 glycosidic linkages and a DP_(n)or DP_(w) of at least 250.

The glucosyltransferase enzyme in certain embodiments of the inventionmay be derived from a Streptococcus species, Leuconostoc species orLactobacillus species, for example. Examples of Streptococcus speciesfrom which the glucosyltransferase may be derived include S. salivarius,S. sobrinus, S. dentirousetti, S. downei, S. mutans, S. oralis, S.gallolyticus and S. sanguinis. Examples of Leuconostoc species fromwhich the glucosyltransferase may be derived include L. mesenteroides,L. amelibiosum, L. argentinum, L. carnosum, L. citreum, L. cremoris, L.dextranicum and L. fructosum. Examples of Lactobacillus species fromwhich the glucosyltransferase may be derived include L. acidophilus, L.delbrueckii, L. helveticus, L. salivarius, L. casei, L. curvatus, L.plantarum, L. sakei, L. brevis, L. buchneri, L. fermentum and L.reuteri.

The glucosyltransferase enzyme herein can comprise, or consist of, anamino acid sequence that is at least 90% identical to the amino acidsequence provided in SEQ ID NO:4, SEQ ID NO:10, SEQ ID NO:12, SEQ IDNO:14, SEQ ID NO:20, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, or SEQ IDNO:34, wherein the glucosyltransferase enzyme has activity.Alternatively, the glucosyltransferase enzyme can comprise, or consistof, an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:4, SEQ ID NO:10, SEQID NO:12, SEQ ID NO:14, SEQ ID NO:20, SEQ ID NO:26, SEQ ID NO:28, SEQ IDNO:30, or SEQ ID NO:34, wherein the glucosyltransferase enzyme hasactivity.

All the amino acid residues disclosed herein at each amino acid positionof the glucosyltransferase enzyme sequences are examples. Given thatcertain amino acids share similar structural and/or charge features witheach other (i.e., conserved), the amino acid at each position in theglucosyltransferase enzyme sequences can be as provided in the disclosedsequences or substituted with a conserved amino acid residue(“conservative amino acid substitution”) as follows:

-   -   1. The following small aliphatic, nonpolar or slightly polar        residues can substitute for each other: Ala (A), Ser (S), Thr        (T), Pro (P), Gly (G);    -   2. The following polar, negatively charged residues and their        amides can substitute for each other: Asp (D), Asn (N), Glu (E),        Gln (Q);    -   3. The following polar, positively charged residues can        substitute for each other: H is (H), Arg (R), Lys (K);    -   4. The following aliphatic, nonpolar residues can substitute for        each other: Ala (A), Leu (L), Ile (I), Val (V), Cys (C), Met        (M); and    -   5. The following large aromatic residues can substitute for each        other: Phe (F), Tyr (Y), Trp (W).

Examples of glucosyltransferase enzymes may be any of the amino acidsequences disclosed herein and that further include 1-300 (or anyinteger there between) residues on the N-terminus and/or C-terminus.Such additional residues may be from a corresponding wild type sequencefrom which the glucosyltransferase enzyme is derived, or may be anothersequence such as an epitope tag (at either N- or C-terminus) or aheterologous signal peptide (at N-terminus), for example. Thus, examplesof glucosyltransferase enzymes include SEQ ID NOs:61, 62, 63 and 64,which represent the wild type sequences from which SEQ ID NOs:30, 4, 28and 20 are derived, respectively.

The glucosyltransferase enzyme can be encoded by the polynucleotidesequence provided in SEQ ID NO:3, SEQ ID NO:9, SEQ ID NO:11, SEQ IDNO:13, SEQ ID NO:19, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, or SEQ IDNO:33, for example. Alternatively, the glucosyltransferase enzyme can beencoded by a polynucleotide sequence that is at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:3, SEQ IDNO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:19, SEQ ID NO:25, SEQ IDNO:27, SEQ ID NO:29, or SEQ ID NO:33.

The glucosyltransferase enzyme in certain embodiments synthesizes polyalpha-1,3-glucan in which at least about 50%, 60%, 70%, 80%, 90%, 95%,96%, 97%, 98%, 99%, or 100% (or any integer between 50% and 100%) of theconstituent glycosidic linkages are alpha-1,3 linkages. In suchembodiments, accordingly, the glucosyltransferase enzyme synthesizespoly alpha-1,3-glucan in which there is less than about 50%, 40%, 30%,20%, 10%, 5%, 4%, 3%, 2%, or 1% of glycosidic linkages that are notalpha-1,3.

In other aspects, the glucosyltransferase enzyme synthesizes polyalpha-1,3-glucan with no branch points or less than about 10%, 9%, 8%,7%, 6%, 5%, 4%, 3%, 2%, or 1% branch points as a percent of theglycosidic linkages in the polymer. Examples of branch points includealpha-1,6 branch points, such as those that are present in mutanpolymer.

The glucosyltransferase enzyme can synthesize poly alpha-1,3-glucanhaving a molecular weight in DP_(n) or DP_(w) of at least about 100.Alternatively, the glucosyltransferase enzyme can synthesize polyalpha-1,3-glucan having a molecular weight in DP_(n) or DP_(w) of atleast about 400. Alternatively still, the glucosyltransferase enzyme cansynthesize poly alpha-1,3-glucan having a molecular weight in DP_(n) orDP_(w) of at least about 100, 150, 200, 250, 300, 350, 400, 450, 500,550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 (or any integerbetween 100 and 1000).

One or more different glucosyltransferase enzymes may be used in thedisclosed invention. The glucosyltransferase enzyme preferably does nothave, or has very little (less than 1%), dextransucrase,reuteransucrase, or alternansucrase activity. The glucosyltransferase incertain embodiments does not comprise amino acid residues 2-1477 of SEQID NO:8 or amino acid residues 138-1477 of SEQ ID NO:8, which arederived from the glucosyltransferase identified in GENBANK under GInumber 47527 (SEQ ID NO:60).

The glucosyltransferase enzyme herein can be primer-independent orprimer-dependent. Primer-independent glucosyltransferase enzymes do notrequire the presence of a primer to perform glucan synthesis. Aprimer-dependent glucosyltransferase enzyme requires the presence of aninitiating molecule in the reaction solution to act as a primer for theenzyme during glucan polymer synthesis. The term “primer” as used hereinrefers to any molecule that can act as the initiator for aglucosyltransferase enzyme. Oligosaccharides and polysaccharides canserve a primers herein, for example. Primers that can be used in certainembodiments include dextran and other carbohydrate-based primers, suchas hydrolyzed glucan, for example. Hydrolyzed glucan can be prepared byacid hydrolysis of a glucan such as poly alpha-glucan. InternationalAppl. Publ. No. WO2013/036918, which is incorporated herein byreference, discloses such preparation of hydrolyzed glucan using polyalpha-1,3-glucan as the starting material. Dextran for use as a primerherein can be dextran T10 (i.e., dextran having a molecular weight of 10kD). Alternatively, the dextran can have a molecular weight of about 2,4, 6, 8, 10, 12, 14, 16, 18, 20, or 25 kD, for example.

The glucosyltransferase enzyme used herein may be produced by any meansknown in the art (e.g., U.S. Pat. No. 7,000,000, which is incorporatedherein by reference). For example, the glucosyltransferase enzyme may beproduced recombinantly in any bacterial (e.g., E. coli such as TOP10,Bacillus sp.) or eukaryotic (e.g., yeasts such as Pichia sp. andSaccharomyces sp.) heterologous gene expression system. Any of theabove-listed nucleic acid sequences can be used for this purpose, forexample.

The glucosyltransferase enzyme used herein may be purified and/orisolated prior to its use, or may be used in the form of a cell lysate,for example. A cell lysate or extract may be prepared from a bacteria(e.g., E. coli) used to heterologously express the enzyme. For example,the bacteria may be subjected to disruption using a French pressure cell(French press). The glucosyltransferase enzyme is soluble in these typeof preparations. The lysate or extract may be used at about 0.15-0.3%(v/v) in a reaction solution for producing poly alpha-1,3-glucan fromsucrose. In certain embodiments, a bacterial cell lysate is firstcleared of insoluble material by means such as centrifugation orfiltration.

In certain embodiments, the heterologous gene expression system may beone that is designed for protein secretion. The glucosyltransferaseenzyme comprises a signal peptide (signal sequence) in such embodiments.The signal peptide may be either its native signal peptide or aheterologous signal peptide.

The activity of the glucosyltransferase enzyme can be determined usingany method known in the art. For example, glucosyltransferase enzymeactivity can be determined by measuring the production of reducingsugars (fructose and glucose) in a reaction solution containing sucrose(50 g/L), dextran T10 (1 mg/mL) and potassium phosphate buffer (pH 6.5,50 mM), where the solution is held at 22-25° C. for 24-30 hours. Thereducing sugars can be measured by adding 0.01 mL of the reactionsolution to a mixture containing 1 N NaOH and 0.1% triphenyltetrazoliumchloride and then monitoring the increase in absorbance at OD_(480nm)for five minutes.

The temperature of the reaction solution herein can be controlled, ifdesired. In certain embodiments, the solution has a temperature betweenabout 5° C. to about 50° C. The temperature of the solution in certainother embodiments is between about 20° C. to about 40° C. Alternatively,the temperature of the solution may be about 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40° C.

The temperature of the reaction solution may be maintained using variousmeans known in the art. For example, the temperature of reactionsolution can be maintained by placing the vessel containing the reactionsolution in an air or water bath incubator set at the desiredtemperature.

The initial concentration of the sucrose in the solution can be about 20g/L to about 400 g/L, for example. Alternatively, the initialconcentration of the sucrose can be about 75 g/L to about 175 g/L, orfrom about 50 g/L to about 150 g/L. Alternatively still, the initialconcentration of the sucrose can be about 40, 50, 60, 70, 80, 90, 100,110, 120, 130, 140, 150, or 160 g/L (or any integer between 40 and 160g/L), for example. The “initial concentration of sucrose” refers to thesucrose concentration in the solution just after all the reactionsolution components have been added (water, sucrose, gtf enzyme).

Sucrose used in the reaction solution can be highly pure 99.5%) or be ofany other purity or grade. For example, the sucrose can have a purity ofat least 99.0%, or be reagent grade sucrose. The sucrose may be derivedfrom any renewable sugar source such as sugar cane, sugar beets,cassava, sweet sorghum, or corn. The sucrose can be provided in any formsuch as crystalline form or non-crystalline form (e.g., syrup or canejuice).

The pH of the reaction solution herein can be between about 4.0 to about8.0. Alternatively, the pH can be about 4.0, 4.5, 5.0, 5.5, 6.0, 6.5,7.0, 7.5, or 8.0. In certain embodiments, the pH of a solutioncontaining water and sucrose may be set before adding theglucosyltransferase enzyme. The pH of the reaction solution can beadjusted or controlled by the addition or incorporation of a suitablebuffer, including but not limited to: phosphate, tris, citrate, or acombination thereof. The concentration of the buffer can be from 0 mM toabout 100 mM, or about 10, 20, or 50 mM, for example. A suitable amountof DTT (dithiothreitol, e.g., about 1.0 mM) can optionally be added tothe reaction solution.

The disclosed invention also concerns a method for producing polyalpha-1,3-glucan comprising the step of contacting at least water,sucrose, and a glucosyltransferase enzyme that synthesizes polyalpha-1,3-glucan. The glucosyltransferase enzyme can comprise an aminoacid sequence that is at least 90% identical to the amino acid sequenceof SEQ ID NO:4, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:20,SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, or SEQ ID NO:34. The polyalpha-1,3-glucan produced in this method can optionally be isolated.

Water, sucrose, and a glucosyltransferase enzyme as described herein arecontacted in a reaction solution. Thus, the method can compriseproviding a reaction solution comprising water, sucrose and aglucosyltransferase enzyme as described herein. It will be understoodthat, as the glucosyltransferase enzyme synthesizes polyalpha-1,3-glucan, the reaction solution becomes a reaction mixture giventhat insoluble poly alpha-1,3-glucan falls out of solution as indicatedby clouding of the reaction. The contacting step of the disclosed methodcan be performed in any number of ways. For example, the desired amountof sucrose can first be dissolved in water (optionally, other componentsmay also be added at this stage of preparation, such as buffercomponents), followed by the addition of the glucosyltransferase enzyme.The solution may be kept still, or agitated via stirring or orbitalshaking, for example. The reaction can be, and typically is, cell-free.

The glucosyltransferase enzyme can optionally be added to water or anaqueous solution (e.g., sucrose in water) that does not contain salt orbuffer when initially preparing the reaction solution. The pH of such apreparation can then be modified as desired, such as to pH 5-6 forexample. The reaction can be carried out to completion without any addedbuffer, if desired.

Completion of the reaction in certain embodiments can be determinedvisually (no more accumulation of precipitated poly alpha-1,3-glucan)and/or by measuring the amount of sucrose left in the solution (residualsucrose), where a percent sucrose consumption of over about 90% canindicate reaction completion. Typically, a reaction of the disclosedprocess will take about 12, 24, 36, 48, 60, 72, 84, or 96 hours tocomplete, depending on certain parameters such as the amount of sucroseand glucosyltransferase enzyme used in the reaction.

The percent sucrose consumption of a reaction in certain embodiments ofthe disclosed process is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%. Alternatively, the percent sucrose consumptionmay be >90% or >95%.

The yield of the poly alpha-1,3-glucan produced in the disclosedinvention can be at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%,14%, 15%, 16%, 17%, 18%, 19%, or 20%, based on the weight of the sucroseused in the reaction solution.

The poly alpha-1,3-glucan produced in the disclosed method mayoptionally be isolated. For example, insoluble poly alpha-1,3-glucan maybe separated by centrifugation or filtration. In doing so, the polyalpha-1,3-glucan is separated from the rest of the reaction solution,which may comprise water, fructose and certain byproducts (e.g.,leucrose, soluble oligosaccharides DP2-DP7). This solution may alsocomprise residual sucrose and glucose monomer.

Poly alpha-1,3 glucan is a potentially low cost polymer which can beenzymatically produced from renewable resources containing sucrose usingglucosyltransferase enzymes. It has been shown that this polymer canform ordered liquid crystalline solutions when the polymer is dissolvedin a solvent under certain conditions (U.S. Pat. No. 7,000,000). Suchsolutions can be spun into continuous, high strength, cotton-likefibers. The poly alpha-1,3-glucan produced using the disclosed inventionhas comparable utilities.

EXAMPLES

The disclosed invention is further defined in the following Examples. Itshould be understood that these Examples, while indicating certainpreferred aspects of the invention, are given by way of illustrationonly. From the above discussion and these Examples, one skilled in theart can ascertain the essential characteristics of this invention, andwithout departing from the spirit and scope thereof, can make variouschanges and modifications of the invention to adapt it to various usesand conditions.

Abbreviations

The meanings of some of the abbreviations used herein are as follows:“g” means gram(s), “h” means hour(s), “mL” means milliliter(s), “psi”means pound(s) per square inch, “wt %” means weight percentage, “μm”means micrometer(s), “° C.” means degrees Celsius, “mg” meansmilligram(s), “mm” means millimeter(s), “μL” means microliter(s), “mmol”means millimole(s), “min” means minute(s), “mol %” means mole percent,“M” means molar, “rpm” means revolutions per minute, “MPa” meansmegaPascals.

General Methods

Preparation of Crude Extracts of Glucosyltransferase (Gtf) Enzymes

Gtf enzymes were prepared as follows. E. coli TOP10® cells (Invitrogen,Carlsbad Calif.) were transformed with a pJexpress404®-based constructcontaining a particular gtf-encoding DNA sequence. Each sequence wascodon-optimized to express the gtf enzyme in E. coli. Individual E. colistrains expressing a particular gtf enzyme were grown in LB (Luriabroth) medium (Becton, Dickinson and Company, Franklin Lakes, N.J.) withampicillin (100 μg/mL) at 37° C. with shaking to OD₆₀₀=0.4-0.5, at whichtime IPTG (isopropyl beta-D-1-thiogalactopyranoside, Cat. No. 16758,Sigma-Aldrich, St. Louis, Mo.) was added to a final concentration of 0.5mM. The cultures were incubated for 2-4 hours at 37° C. following IPTGinduction. Cells were harvested by centrifugation at 5,000×g for 15minutes and resuspended (20% w/v) in 50 mM phosphate buffer pH 7.0supplemented with dithiothreitol (DTT, 1.0 mM). Resuspended cells werepassed through a French Pressure Cell (SLM Instruments, Rochester, N.Y.)twice to ensure >95% cell lysis. Lysed cells were centrifuged for 30minutes at 12,000×g at 4° C. The resulting supernatant was analyzed bythe BCA (bicinchoninic acid) protein assay (Sigma-Aldrich) and SDS-PAGEto confirm expression of the gtf enzyme, and the supernatant was storedat −20° C.

Determination of Gtf Enzymatic Activity

Gtf enzyme activity was confirmed by measuring the production ofreducing sugars (fructose and glucose) in a gtf reaction solution. Areaction solution was prepared by adding a gtf extract (prepared asabove) to a mixture containing sucrose (50 or 150 g/L), potassiumphosphate buffer (pH 6.5, 50 mM), and optionally dextran (1 mg/mL,dextran T10, Cat. No. D9260, Sigma-Aldrich); the gtf extract was addedto 2.5%-5% by volume. The reaction solution was then incubated at 22-25°C. for 24-30 hours, after which it was centrifuged. Supernatant (0.01mL) was added to a mixture containing 1 N NaOH and 0.1%triphenyltetrazolium chloride (Sigma-Aldrich). The mixture was incubatedfor five minutes after which its OD_(480nm) was determined using anULTROSPEC spectrophotometer (Pharmacia LKB, New York, N.Y.) to gauge thepresence of the reducing sugars fructose and glucose.

Determination of Glycosidic Linkages

Glycosidic linkages in the glucan product synthesized by a gtf enzymewere determined by ¹³C NMR (nuclear magnetic resonance). Dry glucanpolymer (25-30 mg) was dissolved in 1 mL of deuterated dimethylsulfoxide (DMSO) containing 3% by weight of LiCl with stirring at 50° C.Using a glass pipet, 0.8 mL of the solution was transferred into a 5-mmNMR tube. A quantitative ¹³C NMR spectrum was acquired using a BrukerAvance 500-MHz NMR spectrometer (Billerica, Mass.) equipped with a CPDULcryoprobe at a spectral frequency of 125.76 MHz, using a spectral windowof 26041.7 Hz. An inverse gated decoupling pulse sequence using waltzdecoupling was used with an acquisition time of 0.629 second, aninter-pulse delay of 5 seconds, and 6000 pulses. The time domain datawas transformed using an exponential multiplication of 2.0 Hz.

Determination of Number Average Degree of Polymerization (DP_(n))

The DP_(n) of a glucan product synthesized by a gtf enzyme wasdetermined by size-exclusion chromatography (SEC). Dry glucan polymerwas dissolved at 5 mg/mL in N,N-dimethyl-acetamide (DMAc) and 5% LiClwith overnight shaking at 100° C. The SEC system used was an Alliance™2695 separation module from Waters Corporation (Milford, Mass.) coupledwith three on-line detectors: a differential refractometer 2410 fromWaters, a multiangle light scattering photometer Heleos™ 8+ from WyattTechnologies (Santa Barbara, Calif.), and a differential capillaryviscometer ViscoStar™ from Wyatt. The columns used for SEC were fourstyrene-divinyl benzene columns from Shodex (Japan) and two linearKD-806M, KD-802 and KD-801 columns to improve resolution at the lowmolecular weight region of a polymer distribution. The mobile phase wasDMAc with 0.11% LiCl. The chromatographic conditions used were 50° C. inthe column and detector compartments, 40° C. in the sample and injectorcompartment, a flow rate of 0.5 mL/min, and an injection volume of 100μL. The software packages used for data reduction were Empower™ version3 from Waters (calibration with broad glucan polymer standard) andAstra® version 6 from Wyatt (triple detection method with columncalibration).

Example 1 Production of Gtf Enzyme 0874 (SEQ ID NO:2)

This Example describes preparing an N-terminally truncated version of aStreptococcus sobrinus gtf enzyme identified in GENBANK under GI number450874 (SEQ ID NO:2, encoded by SEQ ID NO:1; herein referred to as“0874”).

A nucleotide sequence encoding gtf 0874 was synthesized using codonsoptimized for protein expression in E. coli (DNA2.0, Inc., Menlo Park,Calif.). The nucleic acid product (SEQ ID NO:1), encoding gtf 0874 (SEQID NO:2), was subcloned into pJexpress404® (DNA2.0, Inc.) to generatethe plasmid construct identified as pMP57. This plasmid construct wasused to transform E. coli TOP10 cells (Invitrogen, Carlsbad, Calif.) togenerate the strain identified as TOP10/pMP57.

Production of gtf 0874 by bacterial expression and determination of itsenzymatic activity were performed following the procedures disclosed inthe General Methods section. The enzymatic activity of gtf 0874 is shownin Table 2 (see Example 18 below).

Example 2 Production of Gtf Enzyme 6855 (SEQ ID NO:4)

This Example describes preparing an N-terminally truncated version of aStreptococcus salivarius gtf enzyme identified in GENBANK under GInumber 228476855 (SEQ ID NO:4, encoded by SEQ ID NO:3; herein referredto as “6855”).

A nucleotide sequence encoding gtf 6855 was synthesized using codonsoptimized for protein expression in E. coli (DNA2.0, Inc.). The nucleicacid product (SEQ ID NO:3), encoding gtf 6855 (SEQ ID NO:4), wassubcloned into pJexpress404® to generate the plasmid constructidentified as pMP53. This plasmid construct was used to transform E.coli TOP10 cells to generate the strain identified as TOP10/pMP53.

Production of gtf 6855 by bacterial expression and determination of itsenzymatic activity were performed following the procedures disclosed inthe General Methods section. The enzymatic activity of gtf 6855 is shownin Table 2 (see Example 18 below).

Example 3 Production of Gtf Enzyme 2379 (SEQ ID NO:6)

This Example describes preparing an N-terminally truncated version of aStreptococcus salivarius gtf enzyme identified in GENBANK under GInumber 662379 (SEQ ID NO:6, encoded by SEQ ID NO:5; herein referred toas “2379”).

A nucleotide sequence encoding gtf 2379 was synthesized using codonsoptimized for protein expression in E. coli (DNA2.0, Inc.). The nucleicacid product (SEQ ID NO:5), encoding gtf 2379 (SEQ ID NO:6), wassubcloned into pJexpress404® to generate the plasmid constructidentified as pMP66. This plasmid construct was used to transform E.coli TOP10 cells to generate the strain identified as TOP10/pMP66.

Production of gtf 2379 by bacterial expression and determination of itsenzymatic activity were performed following the procedures disclosed inthe General Methods section. The enzymatic activity of gtf 2379 is shownin Table 2 (see Example 18 below).

Example 4 Production of Gtf Enzyme 7527 (GtfJ, SEQ ID NO:8)

This Example describes preparing an N-terminally truncated version of aStreptococcus salivarius gtf enzyme identified in GENBANK under GInumber 47527 (SEQ ID NO:8, encoded by SEQ ID NO:7; herein referred to as“7527” or “GtfJ”).

A nucleotide sequence encoding gtf 7527 was synthesized using codonsoptimized for protein expression in E. coli (DNA2.0, Inc.). The nucleicacid product (SEQ ID NO:7), encoding gtf 7527 (SEQ ID NO:8), wassubcloned into pJexpress404® to generate the plasmid constructidentified as pMP65. This plasmid construct was used to transform E.coli TOP10 cells to generate the strain identified as TOP10/pMP65.

Production of gtf 7527 by bacterial expression and determination of itsenzymatic activity were performed following the procedures disclosed inthe General Methods section. The enzymatic activity of gtf 7527 is shownin Table 2 (see Example 18 below).

Example 5 Production of Gtf Enzyme 1724 (SEQ ID NO:10)

This Example describes preparing an N-terminally truncated version of aStreptococcus downei gtf enzyme identified in GENBANK under GI number121724 (SEQ ID NO:10, encoded by SEQ ID NO:9; herein referred to as“1724”).

A nucleotide sequence encoding gtf 1724 was synthesized using codonsoptimized for protein expression in E. coli (DNA2.0, Inc.). The nucleicacid product (SEQ ID NO:9), encoding gtf 1724 (SEQ ID NO:10), wassubcloned into pJexpress404® to generate the plasmid constructidentified as pMP52. This plasmid construct was used to transform E.coli TOP10 cells to generate the strain identified as TOP10/pMP52.

Production of gtf 1724 by bacterial expression and determination of itsenzymatic activity were performed following the procedures disclosed inthe General Methods section. The enzymatic activity of gtf 1724 is shownin Table 2 (see Example 18 below).

Example 6 Production of Gtf Enzyme 0544 (SEQ ID NO:12)

This Example describes preparing an N-terminally truncated version of aStreptococcus mutans gtf enzyme identified in GENBANK under GI number290580544 (SEQ ID NO:12, encoded by SEQ ID NO:11; herein referred to as“0544”).

A nucleotide sequence encoding gtf 0544 was synthesized using codonsoptimized for protein expression in E. coli (DNA2.0, Inc.). The nucleicacid product (SEQ ID NO:11), encoding gtf 0544 (SEQ ID NO:12), wassubcloned into pJexpress404® to generate the plasmid constructidentified as pMP55. This plasmid construct was used to transform E.coli TOP10 cells to generate the strain identified as TOP10/pMP55.

Production of gtf 0544 by bacterial expression and determination of itsenzymatic activity were performed following the procedures disclosed inthe General Methods section. The enzymatic activity of gtf 0544 is shownin Table 2 (see Example 18 below).

Example 7 Production of Gtf Enzyme 5926 (SEQ ID NO:14)

This Example describes preparing an N-terminally truncated version of aStreptococcus dentirousetti gtf enzyme identified in GENBANK under GInumber 167735926 (SEQ ID NO:14, encoded by SEQ ID NO:13; herein referredto as “5926”).

A nucleotide sequence encoding gtf 5926 was synthesized using codonsoptimized for protein expression in E. coli (DNA2.0, Inc.). The nucleicacid product (SEQ ID NO:13), encoding gtf 5926 (SEQ ID NO:14), wassubcloned into pJexpress404® to generate the plasmid constructidentified as pMP67. This plasmid construct was used to transform E.coli TOP10 cells to generate the strain identified as TOP10/pMP67.

Production of gtf 5926 by bacterial expression and determination of itsenzymatic activity were performed following the procedures disclosed inthe General Methods section. The enzymatic activity of gtf 5926 is shownin Table 2 (see Example 18 below).

Example 8 Production of Gtf Enzyme 4297 (SEQ ID NO:16)

This Example describes preparing an N-terminally truncated version of aStreptococcus oralis gtf enzyme identified in GENBANK under GI number7684297 (SEQ ID NO:16, encoded by SEQ ID NO:15; herein referred to as“4297”).

A nucleotide sequence encoding gtf 4297 was synthesized using codonsoptimized for protein expression in E. coli (DNA2.0, Inc.). The nucleicacid product (SEQ ID NO:15), encoding gtf 4297 (SEQ ID NO:16), wassubcloned into pJexpress404® to generate the plasmid constructidentified as pMP62. This plasmid construct was used to transform E.coli TOP10 cells to generate the strain identified as TOP10/pMP62.

Production of gtf 4297 by bacterial expression and determination of itsenzymatic activity were performed following the procedures disclosed inthe General Methods section. The enzymatic activity of gtf 4297 is shownin Table 2 (see Example 18 below).

Example 9 Production of Gtf Enzyme 5618 (SEQ ID NO:18)

This Example describes preparing an N-terminally truncated version of aStreptococcus sanguinis gtf enzyme identified in GENBANK under GI number328945618 (SEQ ID NO:18, encoded by SEQ ID NO:17; herein referred to as“5618”).

A nucleotide sequence encoding gtf 5618 was synthesized using codonsoptimized for protein expression in E. coli (DNA2.0, Inc.). The nucleicacid product (SEQ ID NO:17), encoding gtf 5618 (SEQ ID NO:18), wassubcloned into pJexpress404® to generate the plasmid constructidentified as pMP56. This plasmid construct was used to transform E.coli TOP10 cells to generate the strain identified as TOP10/pMP56.

Production of gtf 5618 by bacterial expression and determination of itsenzymatic activity were performed following the procedures disclosed inthe General Methods section. The enzymatic activity of gtf 5618 is shownin Table 2 (see Example 18 below).

Example 10 Production of Gtf Enzyme 2765 (SEQ ID NO:20)

This Example describes preparing an N-terminally truncated version of aStreptococcus sp. gtf enzyme identified in GENBANK under GI number322372765 (SEQ ID NO:20, encoded by SEQ ID NO:19; herein referred to as“2765”).

A nucleotide sequence encoding gtf 2765 was synthesized using codonsoptimized for protein expression in E. coli (DNA2.0, Inc.). The nucleicacid product (SEQ ID NO:19), encoding gtf 2765 (SEQ ID NO:20), wassubcloned into pJexpress404® to generate the plasmid constructidentified as pMP73. This plasmid construct was used to transform E.coli TOP10 cells to generate the strain identified as TOP10/pMP73.

Production of gtf 2765 by bacterial expression and determination of itsenzymatic activity were performed following the procedures disclosed inthe General Methods section. The enzymatic activity of gtf 2765 is shownin Table 2 (see Example 18 below).

Example 11 Production of Gtf Enzyme 4700 (SEQ ID NO:22)

This Example describes preparing an N-terminally truncated version of aLeuconostoc mesenteroides gtf enzyme identified in GENBANK under GInumber 21654700 (SEQ ID NO:22, encoded by SEQ ID NO:21; herein referredto as “4700”).

A nucleotide sequence encoding gtf 2765 was synthesized using codonsoptimized for protein expression in E. coli (DNA2.0, Inc.). The nucleicacid product (SEQ ID NO:21), encoding gtf 4700 (SEQ ID NO:22), wassubcloned into pJexpress404® to generate the plasmid constructidentified as pMP83. This plasmid construct was used to transform E.coli TOP10 cells to generate the strain identified as TOP10/pMP83.

Production of gtf 4700 by bacterial expression and determination of itsenzymatic activity were performed following the procedures disclosed inthe General Methods section. The enzymatic activity of gtf 4700 is shownin Table 2 (see Example 18 below).

Example 12 Production of Gtf Enzyme 1366 (SEQ ID NO:24)

This Example describes preparing an N-terminally truncated version of aStreptococcus criceti gtf enzyme identified in GENBANK under GI number146741366 (SEQ ID NO:24, encoded by SEQ ID NO:23; herein referred to as“1366”).

A nucleotide sequence encoding gtf 1366 was synthesized using codonsoptimized for protein expression in E. coli (DNA2.0, Inc.). The nucleicacid product (SEQ ID NO:23), encoding gtf 1366 (SEQ ID NO:24), wassubcloned into pJexpress404® to generate the plasmid constructidentified as pMP86. This plasmid construct was used to transform E.coli TOP10 cells to generate the strain identified as TOP10/pMP86.

Production of gtf 1366 by bacterial expression and determination of itsenzymatic activity were performed following the procedures disclosed inthe General Methods section. The enzymatic activity of gtf 1366 is shownin Table 2 (see Example 18 below).

Example 13 Production of Gtf Enzyme 0427 (SEQ ID NO:26)

This Example describes preparing an N-terminally truncated version of aStreptococcus sobrinus gtf enzyme identified in GENBANK under GI number940427 (SEQ ID NO:26, encoded by SEQ ID NO:25; herein referred to as“0427”).

A nucleotide sequence encoding gtf 0427 was synthesized using codonsoptimized for protein expression in E. coli (DNA2.0, Inc.). The nucleicacid product (SEQ ID NO:25), encoding gtf 0427 (SEQ ID NO:26), wassubcloned into pJexpress404® to generate the plasmid constructidentified as pMP87. This plasmid construct was used to transform E.coli TOP10 cells to generate the strain identified as TOP10/pMP87.

Production of gtf 0427 by bacterial expression and determination of itsenzymatic activity were performed following the procedures disclosed inthe General Methods section. The enzymatic activity of gtf 0427 is shownin Table 2 (see Example 18 below).

Example 14 Production of Gtf Enzyme 2919 (SEQ ID NO:28)

This Example describes preparing an N-terminally truncated version of aStreptococcus salivarius gtf enzyme identified in GENBANK under GInumber 383282919 (SEQ ID NO:28, encoded by SEQ ID NO:27; herein referredto as “2919”).

A nucleotide sequence encoding gtf 2919 was synthesized using codonsoptimized for protein expression in E. coli (DNA2.0, Inc.). The nucleicacid product (SEQ ID NO:27), encoding gtf 2919 (SEQ ID NO:28), wassubcloned into pJexpress404® to generate the plasmid constructidentified as pMP88. This plasmid construct was used to transform E.coli TOP10 cells to generate the strain identified as TOP10/pMP88.

Production of gtf 2919 by bacterial expression and determination of itsenzymatic activity were performed following the procedures disclosed inthe General Methods section. The enzymatic activity of gtf 2919 is shownin Table 2 (see Example 18 below).

Example 15 Production of Gtf Enzyme 2678 (SEQ ID NO:30)

This Example describes preparing an N-terminally truncated version of aStreptococcus salivarius gtf enzyme identified in GENBANK under GInumber 400182678 (SEQ ID NO:30 encoded by SEQ ID NO:29; herein referredto as “2678”).

A nucleotide sequence encoding gtf 2678 was synthesized using codonsoptimized for protein expression in E. coli (DNA2.0, Inc.). The nucleicacid product (SEQ ID NO:29), encoding gtf 2678 (SEQ ID NO:30), wassubcloned into pJexpress404® to generate the plasmid constructidentified as pMP89. This plasmid construct was used to transform E.coli TOP10 cells to generate the strain identified as TOP10/pMP89.

Production of gtf 2678 by bacterial expression and determination of itsenzymatic activity were performed following the procedures disclosed inthe General Methods section. The enzymatic activity of gtf 2678 is shownin Table 2 (see Example 18 below).

Example 16 Production of Gtf Enzyme 2381 (SEQ ID NO:32)

This Example describes preparing an N-terminally truncated version of aStreptococcus salivarius gtf enzyme identified in GENBANK under GInumber 662381 (SEQ ID NO:32 encoded by SEQ ID NO:31; herein referred toas “2381”).

A nucleotide sequence encoding gtf 2381 was synthesized using codonsoptimized for protein expression in E. coli (DNA2.0, Inc.). The nucleicacid product (SEQ ID NO:31), encoding gtf 2381 (SEQ ID NO:32), wassubcloned into pJexpress404® to generate the plasmid constructidentified as pMP96. This plasmid construct was used to transform E.coli TOP10 cells to generate the strain identified as TOP10/pMP96.

Production of gtf 2381 by bacterial expression and determination of itsenzymatic activity were performed following the procedures disclosed inthe General Methods section. The enzymatic activity of gtf 2381 is shownin Table 2 (see Example 18 below).

Example 17 Production of Gtf Enzyme 3929 (SEQ ID NO:34) and AdditionalGtf Enzymes

This Example describes preparing an N-terminally truncated version of aStreptococcus salivarius gtf enzyme identified in GENBANK under GInumber 387783929 (SEQ ID NO:34 encoded by SEQ ID NO:33; herein referredto as “3929”).

A nucleotide sequence encoding gtf 3929 was synthesized using codonsoptimized for protein expression in E. coli (DNA2.0, Inc.). The nucleicacid product (SEQ ID NO:33), encoding gtf 3929 (SEQ ID NO:34), wassubcloned into pJexpress404® to generate the plasmid constructidentified as pMP97. This plasmid construct was used to transform E.coli TOP10 cells to generate the strain identified as TOP10/pMP97.

Production of gtf 3929 by bacterial expression and determination of itsenzymatic activity were performed following the procedures disclosed inthe General Methods section. The enzymatic activity of gtf 3929 is shownin Table 2 (see Example 18 below).

Additional gtf enzymes were produced in a similar manner. Briefly,N-terminally truncated versions of enzymes identified in GENBANK underGI numbers 228476907 (a Streptococcus salivarius gtf, SEQ ID NO:36,herein referred to as “6907”), 228476661 (a Streptococcus salivariusgtf, SEQ ID NO:38, herein referred to as “6661”), 334280339 (aStreptococcus gallolyticus gtf, SEQ ID NO:40, herein referred to as“0339”), 3130088 (a Streptococcus mutans gtf, SEQ ID NO:42, hereinreferred to as “0088”), 24379358 (a Streptococcus mutans gtf, SEQ IDNO:44, herein referred to as “9358”), 325978242 (a Streptococcusgallolyticus gtf, SEQ ID NO:46, herein referred to as “8242”), 324993442(a Streptococcus sanguinis gtf, SEQ ID NO:48, herein referred to as“3442”), 47528 (a Streptococcus salivarius gtf, SEQ ID NO:50, hereinreferred to as “7528”), 322373279 (a Streptococcus sp. gtf, SEQ IDNO:52, herein referred to as “3279”), 170016491 (a Leuconostoc citreumgtf, SEQ ID NO:54, herein referred to as “6491”), 228476889 (aStreptococcus salivarius gtf, SEQ ID NO:56, herein referred to as“6889”), 51574154 (a Lactobacillus reuteri gtf, SEQ ID NO:58, hereinreferred to as “4154”), and 322373298 (a Streptococcus sp. gtf, SEQ IDNO:59, herein referred to as “3298”) were prepared and tested forenzymatic activity (Table 2, see Example 18 below).

Example 18 Production of Insoluble Glucan Polymer with Gtf Enzymes

This Example describes using the gtf enzymes prepared in the aboveExamples to synthesize glucan polymer.

Reactions were performed with each of the above gtf enzymes followingthe procedures disclosed in the General Methods section. Briefly, gtfreaction solutions were prepared comprising sucrose (50 g/L), potassiumphosphate buffer (pH 6.5, 50 mM) and a gtf enzyme (2.5% extract byvolume). After 24-30 hours at 22-25° C., insoluble glucan polymerproduct was harvested by centrifugation, washed three times with water,washed once with ethanol, and dried at 50° C. for 24-30 hours.

Following the procedures disclosed in the General Methods section, theglycosidic linkages in the insoluble glucan polymer product from eachreaction were determined by ¹³C NMR, and the DP_(n) for each product wasdetermined by SEC. The results of these analyses are shown in Table 2.

TABLE 2 Linkages and DP_(n) of Glucan Produced by Various Gtf EnzymesReducing Insoluble Glucan Alpha SEQ ID Sugars Glucan Linkages Gtf NO.Produced? Produced? % 1, 3 % 1, 6 DP_(n) 0874 2 yes yes 100 0 60 6855 4yes yes 100 0 440 2379 6 yes yes 37 63 310 7527 8 yes yes 100 0 440 172410 yes yes 100 0 250 0544 12 yes yes 62 36 980 5926 14 yes yes 100 0 2604297 16 yes yes 31 67 800 5618 18 yes yes 34 66 1020 2765 20 yes yes 1000 280 4700 22 yes no 1366 24 yes no 0427 26 yes yes 100 0 120 2919 28yes yes 100 0 250 2678 30 yes yes 100 0 390 2381 32 yes no 3929 34 yesyes 100 0 280 6907 36 yes no 6661 38 yes no 0339 40 yes no 0088 42 yesno 9358 44 yes no 8242 46 yes no 3442 48 yes no 7528 50 yes no 3279 52yes no 6491 54 yes no 6889 56 yes no 4154 58 yes no 3298 59 yes no nonena no no

Several gtf enzymes produced insoluble glucan products (Table 2).However, only gtf enzymes 6855 (SEQ ID NO:4), 7527 (gtfJ, SEQ ID NO:8),1724 (SEQ ID NO:10), 0544 (SEQ ID NO:12), 5926 (SEQ ID NO:14), 2765 (SEQID NO:20), 0427 (SEQ ID NO:26), 2919 (SEQ ID NO:28), 2678 (SEQ IDNO:30), and 3929 (SEQ ID NO:34) produced glucan comprising at least 50%alpha-1,3 linkages and having a DP_(n) of at least 100. These enzymesare therefore suitable for producing glucan polymers for fiberapplications.

Only gtfs 6855 (SEQ ID NO:4), 7527 (gtfJ, SEQ ID NO:8), 1724 (SEQ IDNO:10), 5926 (SEQ ID NO:14), 2765 (SEQ ID NO:20), 0427 (SEQ ID NO:26),2919 (SEQ ID NO:28), 2678 (SEQ ID NO:30), and 3929 (SEQ ID NO:34)produced glucan polymer comprising 100% alpha-1,3 linkages and having aDP_(n) of at least 100. These results, in which only nine out of thirtygtfs were able to produce glucan with 100% alpha-1,3 linkages and aDP_(n) of at least 100, indicate that not all gtf enzymes are capable ofproducing high molecular weight, insoluble glucan with a high level ofalpha-1,3 linkages. Fewer gtf enzymes were able to produce glucanpolymer comprising 100% alpha-1,3 linkages and having a DP_(n) of atleast 250.

Thus, gtf enzymes capable of producing glucan polymer comprising 100%alpha-1,3 linkages and a DP_(n) of at least 100 were identified. Theseenzymes can be used to produce glucan suitable for producing fibers.

What is claimed is:
 1. A reaction solution comprising water, sucrose anda glucosyltransferase enzyme that synthesizes poly alpha-1,3-glucan,wherein said glucosyltransferase enzyme consists of an amino acidsequence that is at least 93% identical to SEQ ID NO:10.
 2. The reactionsolution of claim 1, wherein said glucosyltransferase enzyme synthesizespoly alpha-1,3-glucan having at least 50% alpha-1,3 glycosidic linkagesand a number average degree of polymerization of at least
 100. 3. Thereaction solution of claim 2, wherein said glucosyltransferase enzymesynthesizes poly alpha-1,3-glucan having 100% alpha-1,3 glycosidiclinkages and a number average degree of polymerization of at least 100.4. The reaction solution of claim 3, wherein said glucosyltransferaseenzyme synthesizes poly alpha-1,3-glucan having 100% alpha-1,3glycosidic linkages and a number average degree of polymerization of atleast
 250. 5. The reaction solution of claim 1, further comprising aprimer.
 6. The reaction solution of claim 5, wherein the primer isdextran.
 7. The reaction solution of claim 5, wherein the primer ishydrolyzed glucan.
 8. The reaction solution of claim 1, wherein saidglucosyltransferase enzyme consists of an amino acid sequence that is atleast 95% identical to SEQ ID NO:10.
 9. The reaction solution of claim8, wherein said glucosyltransferase enzyme consists of an amino acidsequence that is at least 97% identical to SEQ ID NO:10.
 10. Thereaction solution of claim 9, wherein said glucosyltransferase enzymeconsists of an amino acid sequence that is at least 99% identical to SEQID NO:10.
 11. The reaction solution of claim 1, wherein a heterologousamino acid sequence of 1-300 residues is at the N-terminus and/orC-terminus of said glucosyltransferase enzyme.
 12. A method forproducing poly alpha-1,3-glucan comprising: a) contacting at leastwater, sucrose, and a glucosyltransferase enzyme that synthesizes polyalpha-1,3-glucan, wherein said glucosyltransferase enzyme consists of anamino acid sequence that is at least 93% identical to SEQ ID NO:10;whereby poly alpha-1,3-glucan is produced; and b) optionally, isolatingthe poly alpha-1,3-glucan produced in step (a).
 13. The method of claim12, wherein said glucosyltransferase enzyme synthesizes polyalpha-1,3-glucan having at least 50% alpha-1,3 glycosidic linkages and anumber average degree of polymerization of at least
 100. 14. The methodof claim 13, wherein said glucosyltransferase enzyme synthesizes polyalpha-1,3-glucan having 100% alpha-1,3 glycosidic linkages and a numberaverage degree of polymerization of at least
 100. 15. The method ofclaim 14, wherein said glucosyltransferase enzyme synthesizes polyalpha-1,3-glucan having 100% alpha-1,3 glycosidic linkages and a numberaverage degree of polymerization of at least
 250. 16. The method ofclaim 12, wherein step (a) further comprises contacting a primer withthe water, sucrose, and glucosyltransferase enzyme.
 17. The method ofclaim 16, wherein the primer is dextran.
 18. The method of claim 16,wherein the primer is hydrolyzed glucan.
 19. The method of claim 12,wherein said glucosyltransferase enzyme consists of an amino acidsequence that is at least 95% identical to SEQ ID NO:10.
 20. The methodof claim 19, wherein said glucosyltransferase enzyme consists of anamino acid sequence that is at least 97% identical to SEQ ID NO:10. 21.The method of claim 20, wherein said glucosyltransferase enzyme consistsof an amino acid sequence that is at least 99% identical to SEQ IDNO:10.
 22. The method of claim 12, wherein a heterologous amino acidsequence of 1-300 residues is at the N-terminus and/or C-terminus ofsaid glucosyltransferase enzyme.